Mycobacterium tuberculosis (MTB) promotes its survival by preventing phagosome maturation. Reversing this process by targeting host proteins or pathways provides a potential future therapeutic approach to eliminate intracellular MTB. We propose a novel, alternative approach to identify critical host components of the virulent MTB H37Rv phagosome maturation pathway in human macrophages. [PARAGRAPH] Phagosome maturation is mediated by the recruitment or elimination of select host proteins from phagosomes. Therefore, host proteins that mediate this process are differentially expressed on mature versus immature phagosomes. While conventional techniques have identified a few of these proteins, we will systematically identify a set of host proteins that are differentially expressed on mature versus immature virulent MTB H37Rv phagosomes in human macrophages by state-of-the-art proteomic technologies. Of these differentially expressed host proteins, those that specifically inhibit MTB phagosome maturation will be identified through inhibition of expression. We will additionally assess the impact of inhibiting previously identified, differentially expressed host proteins, on MTB H37Rv phagosome maturation and viability in human macrophages, since many of these studies have been limited to avirulent mycobacterial strains in mouse macrophages. [PARAGRAPH] AIM 1: Identify differentially expressed host proteins in live and dead MTB H37Rv phagosomes by proteomic analysis of phagosomes isolated from a human monocyte cell line, THP-1. Validate our findings by Western blot analysis of live and dead MTB H37Rv phagosomes. We have previously standardized the isolation of MTB phagosomes in murine (Ramachandra et al., J. Exp. Med. 2001 (1)) and human macrophages (2). AIM 2: Inhibit expression of a select number of newly identified, differentially expressed host proteins to define their impact on MTB H37Rv phagosome maturation and viability in THP-1 macrophages. We will additionally analyze impact of inhibiting expression of Rab 5, Rab 14 and Rab22a on MTB H37Rv phagosome maturation. [PARAGRAPH] Aim 1 will be the focus of year one. Aim 2 will be the focus of year 1 and 2. We predict that the data generated will lay the foundation for future studies to identify novel drug targets that promote MTB phagosome maturation and killing with a lower risk of drug resistance. PUBLIC HEALTH RELEVANCE: The capacity of Mycobacterium tuberculosis (MTB) to latently infect over one billion people and cause two million fatalities annually rests with its ability to block phagosomal maturation into the phagolysosome. Agents that target host proteins/pathways and promote MTB phagosome maturation and killing will provide a novel way to eliminate intracellular MTB and also make drug resistance more difficult to achieve. We propose to use proteomics as a novel, rapid, comprehensive, unbiased and data-driven tool to identify host proteins that may serve as potential therapeutic targets by promoting virulent MTB phagosome maturation and killing.